Lixuan Zhan

Activation of Akt/FoxO signaling pathway contributes to induction of neuroprotection against transient global cerebral ischemia by hypoxic pre‐conditioning in adult rats

J. Neurochem. (2010) 114, 897–908.

Hypoxia-inducible factor 1α mediates neuroprotection of hypoxic postconditioning against global cerebral ischemia.

Abstract Hypoxia administered after transient global cerebral ischemia (tGCI) has been shown to induce neuroprotection in adult rats, but the underlying mechanisms for this protection are unclear. Here, we tested the hypothesis that hypoxic postconditioning (HPC) induces neuroprotection through upregulation of hypoxia-inducible factor 1&agr; (HIF-1&agr;) and vascular endothelial growth factor (VEGF), and that this involves phosphatidylinositol-3-kinase (PI3K), p38 mitogen–activated protein kinase (p38 MAPK), and mitogen-activated protein kinase/extracellular signal–regulated kinase kinase (MEK) pathways. The expression of HIF-1&agr;, VEGF, and cleaved caspase-9 were determined by immunohistochemistry and Western blot. As pharmacologic interventions, the HIF-1&agr; inhibitor 2-methoxyestradiol (2ME2), PI3K inhibitor LY294002, p38 MAPK inhibitor SB203580, and MEK inhibitor U0126 were administered before HPC or after tGCI. We found that HPC maintained the higher expression of HIF-1&agr; and VEGF and decreased cleaved caspase-9 levels in CA1 after tGCI. These effects were reversed by 2ME2 administered before HPC, and the neuroprotection of HPC was abolished. LY294002 and SB203580 decreased the expression of HIF-1&agr; and VEGF after HPC, whereas U0126 increased HIF-1&agr; and VEGF after tGCI. These findings suggested that HIF-1&agr; exerts neuroprotection induced by HPC against tGCI through VEGF upregulation and cleaved caspase-9 downregulation, and that the PI3K, p38 MAPK, and MEK pathways are involved in the regulation of HIF-1&agr; and VEGF.

Association of transforming growth factor-β1 gene C-509T and T869C polymorphisms with atherosclerotic cerebral infarction in the Chinese: a case-control study

BackgroundTransforming growth factor-β1 (TGF-β1) is a multifunctional cytokine involved in inflammation and pathogenesis of atherosclerosis. There is scant information on the relation between variations within the TGF-β1 gene polymorphisms and risks of ischemic cerebrovascular diseases. Therefore, this case-controlled study was carried out to investigate the possible association of the TGF-β1 gene C-509T and T869C polymorphisms, and their combined genotypes with the risk of atherosclerotic cerebral infarction (CI) in the Chinese population.ResultsWe recruited 164 CI patients and 167 healthy control subjects who were frequency-matched for age and gender. The frequencies of the -509TT genotype and T allele gene were significantly higher in the CI group (P = 0.007, P = 0.006). The frequencies of +869CC genotype and C allele were higher in the CI group (P = 0.002, P = 0.004). In the CI group, the individuals with -509TT genotype had a significantly higher level of plasma triglyceride (TG) (P = 0.017). +869CC genotype correlated significantly with higher level of plasma low density lipoprotein cholesterol (LDL-c) in the CI group (P = 0.015). With haplotype analysis, the frequency of the -509T/+869C combined genotype was significantly higher in the CI group than in controls (P < 0.001).ConclusionsOur study suggests that C-509T and T869C gene polymorphisms in TGF-β1 may be a critical risk factor of genetic susceptibility to CI in the Chinese population.

Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase.

We demonstrated previously that 30min of hypoxic preconditioning (HPC) applied 1day before 10min of transient global cerebral ischemia (tGCI) reduced neuronal loss in the hippocampal CA1 subregion in adult rats. The aim of the present study was to investigate the role of Na(+)/K(+)-ATPase and protein kinase Mζ (PKMζ) in the protective effect of HPC against tGCI in adult rats. We found that the activity of Na(+)/K(+)-ATPase decreased in the hippocampal CA1 subregion after 10min of tGCI. This effect was not seen after 30min of HPC in adult rats. Corresponding to the changes in Na(+)/K(+)-ATPase activity, the surface expression of Na(+)/K(+)-ATPase α1 subunit increased after HPC. Furthermore, HPC dramatically reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the hippocampal CA1 subregion after tGCI. However, neither PKMζ nor phosphorylation of PKMζ was changed after tGCI or HPC. The results of the present study are consistent with the hypothesis that both enhanced recovery of Na(+)/K(+)-ATPase activity due to preserved the protein levels of Na(+)/K(+)-ATPase α1 subunit and reduced DNA fragmentation after tGCI contribute to the protection afforded by HPC. However, PKMζ activation does not appear to play a role in this neuroprotection.

Autophagosome maturation mediated by Rab7 contributes to neuroprotection of hypoxic preconditioning against global cerebral ischemia in rats

Autophagy disruption leads to neuronal damage in hypoxic–ischemic brain injury. Rab7, a member of the Rab GTPase superfamily, has a unique role in the regulation of autophagy. Hypoxic preconditioning (HPC) provides neuroprotection against transient global cerebral ischemia (tGCI). However, the underlying mechanisms remain poorly understood. Thus, the current study explored the potential molecular mechanism of the neuroprotective effect of HPC by investigating how Rab7 mediates autophagosome (AP) maturation after tGCI in adult rats. We found that HPC attenuated AP accumulation in the hippocampal CA1 region after tGCI via restoration of autophagic flux. We also confirmed that this HPC-induced neuroprotection was not caused by the increase in lysosomes or the improvement of lysosomal function after tGCI. Electron microscopic analysis then revealed an increase in autolysosomes in CA1 neurons of HPC rats. Moreover, the inhibition of autophagosome-lysosome fusion by chloroquine significantly aggravated neuronal death in CA1, indicating that AP maturation contributes to HPC-induced neuroprotection against neuronal injury after tGCI. Furthermore, the activation of Rab7 was found to be involved in the neuroprotective effect of AP maturation after HPC. At last, the knockdown of ultraviolet radiation resistance-associated gene (UVRAG) in vivo disrupted the interaction between Vps16 and Rab7, attenuated the activation of Rab7, interrupted autophagic flux, and ultimately abrogated the HPC-induced neuroprotection against tGCI. Our results indicated that AP maturation was enhanced by the activation of Rab7 via UVRAG-Vps16 interaction, which further demonstrated the potential neuroprotective role of Rab7 in HPC against tGCI-induced neuronal injury in adult rats.

Rab7 may be a novel therapeutic target for neurologic diseases as a key regulator in autophagy

Macroautophagy is an evolutionally conserved membrane trafficking pathway that delivers intracellular materials to lysosomes for degradation and recycling. Rab7, as a member of the Rab GTPase superfamily, has a unique role in the regulation of macroautophagy, especially in modulating autophagy flux. The functional states of Rab7 generally switch between GTP‐bound and GDP‐bound states under the control of guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Activated GTP‐Rab7 is capable of regulating autophagosome formation, autophagosome transportation along microtubules, endosome and autophagosome maturation, as well as lysosome biogenesis via interacting with its effector molecules. Rab7‐mediated macroautophagy is closely associated with various pathological processes of several neurologic diseases, such as Parkinsons disease, Huntingtons disease, Alzheimers disease, Charcot‐Marie‐Tooth type 2B disease, and cerebral ischemic diseases. Considering that macroautophagy can be the prime therapeutic target in certain nervous system diseases, in‐depth study of Rab7 in the regulation of macroautophagy may be helpful to identify novel strategies for the treatment of autophagy‐related neurologic diseases.

Neuroprotection of Hypoxic Postconditioning against Global Cerebral Ischemia through Influencing Posttranslational Regulations of Heat Shock Protein 27 in Adult Rats

We previously reported that hypoxic postconditioning (HPC) ameliorated hippocampal neuronal death induced by transient global cerebral ischemia (tGCI) in adult rats. However, the mechanism of HPC‐induced neuroprotection is still elusive. Notably, heat shock protein 27 (Hsp27) has recently emerged as a potent neuroprotectant in cerebral ischemia. Although its robust protective effect on stroke has been recognized, the mechanism of Hsp27‐mediated neuroprotection is largely unknown. Here, we investigated the potential molecular mechanism by which HPC modulates the posttranslational regulations of Hsp27 after tGCI. We found that HPC increased expression of Hsp27 in CA1 subregion after tGCI. Inhibition of Hsp27 expression with lentivirus‐mediated short hairpin RNA (shRNA) abolished the neuroprotection induced by HPC in vivo. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor, resulted in a significant decrease in the degradation rate of Hsp27 protein in postconditioned rats, suggesting that the increase in the expression of Hsp27 after HPC might result from its decreased degradation. Next, pretreatment with leupeptin, a lysosomal inhibitor, resulted in an accumulation of Hsp27 after tGCI, indicating that autophagic pathway may be responsible for the degradation of Hsp27. We further showed that the formation of LC3‐II and autophagosomes increased after tGCI. Meanwhile, the degradation of Hsp27 was suppressed and neuronal damage was reduced when blocking autophagy with 3‐Methyladenine, whereas activating autophagy with rapamycin showed an opposite tendency. Lastly, we confirmed that HPC increased the expression of phosphorylated MAPKAP kinase 2 (MK2) and Hsp27 after tGCI. Also, administration of SB203580, a p38 mitogen‐activated protein kinase inhibitor, decreased the expressions of phosphorylated MK2 and Hsp27. Our results suggested that inhibition of Hsp27 degradation mediated by down‐regulation of autophagy may induce ischemic tolerance after HPC. Additionally, phosphorylation of Hsp27 induced by MK2 might be associated with the neuroprotection of HPC.

Inhibition of Cathepsin B Alleviates Secondary Degeneration in Ipsilateral Thalamus After Focal Cerebral Infarction in Adult Rats

Secondary degeneration in areas beyond ischemic foci can inhibit poststroke recovery. The cysteine protease Cathepsin B (CathB) regulates cell death and intracellular protein catabolism. To investigate the roles of CathB in the development of secondary degeneration in the ventroposterior nucleus (VPN) of the ipsilateral thalamus after focal cerebral infarction, infarct volumes, immunohistochemistry and immunofluorescence, and Western blotting analyses were conducted in a distal middle cerebral artery occlusion (dMCAO) stroke model in adult rats. We observed marked neuron loss and gliosis in the ipsilateral thalamus after dMCAO, and the expression of CathB and cleaved caspase-3 in the VPN was significantly upregulated; glial cells were the major source of CathB. Although it had no effect on infarct volume, delayed intracerebroventricular treatment with the membrane-permeable CathB inhibitor CA-074Me suppressed the expression of CathB and cleaved caspase-3 in ipsilateral VPN and accordingly alleviated the secondary degeneration. These data indicate that CathB mediates a novel mechanism of secondary degeneration in the VPN of the ipsilateral thalamus after focal cortical infarction and suggest that CathB might be a therapeutic target for the prevention of secondary degeneration in patients after stroke.

Inhibition of Cathepsins B Induces Neuroprotection Against Secondary Degeneration in Ipsilateral Substantia Nigra After Focal Cortical Infarction in Adult Male Rats

Stroke is the leading cause of adult disability in the world. In general, recovery from stroke is incomplete. Accumulating evidences have shown that focal cerebral infarction leads to dynamic trans-neuronal degeneration in non-ischemic remote brain regions, with the disruption of connections to synapsed neurons sustaining ischemic insults. Previously, we had reported that the ipsilateral striatum, thalamus degenerated in succession after permanent distal branch of middle cerebral artery occlusion (dMCAO) in Sprague-Dawley (SD) rats and cathepsin (Cath) B was activated before these relay degeneration. Here, we investigate the role of CathB in the secondary degeneration of ipsilateral substantia nigra (SN) after focal cortical infarction. We further examined whether the inhibition of CathB with L-3-trans-(Propyl-carbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA-074Me) would attenuate secondary degeneration through enhancing the cortico-striatum-nigral connections and contribute to the neuroprotective effects. Our results demonstrated that secondary degeneration in the ipsilateral SN occurred and CathB was upregulated in the ipsilateral SN after focal cortical infarction. The inhibition of CathB with CA-074Me reduced the neuronal loss and gliosis in the ipsilateral SN. Using biotinylated dextran amine (BDA) or pseudorabies virus (PRV) 152 as anterograde or retrograde tracer to trace striatum-nigral and cortico-nigral projections pathway, CA-074Me can effectively enhance the cortico-striatum-nigral connections and exert neuroprotection against secondary degeneration in the ipsilateral SN after cortical ischemia. Our study suggests that the lysosomal protease CathB mediates the secondary damage in the ipsilateral SN after dMCAO, thus it can be a promising neuroprotective target for the rehabilitation of stroke patients.

Hypoxic Preconditioning Maintains GLT-1 Against Transient Global Cerebral Ischemia Through Upregulating Cx43 and Inhibiting c-Src

Transient global cerebral ischemia (tGCI) causes excessive release of glutamate from neurons. Astrocytic glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) together play a predominant role in maintaining glutamate at normal extracellular concentrations. Though our previous studies reported the alleviation of tGCI-induced neuronal death by hypoxic preconditioning (HPC) in hippocampal Cornu Ammonis 1 (CA1) of adult rats, the underlying mechanism has not yet been fully elaborated. In this study, we aimed to investigate the roles of GLT-1 and GS in the neuroprotection mediated by HPC against tGCI and to ascertain whether these roles can be regulated by connexin 43 (Cx43) and cellular-Src (c-Src) activity. We found that HPC decreased the level of extracellular glutamate in CA1 after tGCI via maintenance of GLT-1 expression and GS activity. Inhibition of GLT-1 expression with dihydrokainate (DHK) or inhibition of GS activity with methionine sulfoximine (MSO) abolished the neuroprotection induced by HPC. Also, HPC markedly upregulated Cx43 and inhibited p-c-Src expression in CA1 after tGCI, whereas inhibition of Cx43 with Gap26 dramatically reversed this effect. Furthermore, inhibition of p-c-Src with 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) decreased c-Src activity, increased protein levels of GLT-1 and Cx43, enhanced GS activity, and thus reduced extracellular glutamate level in CA1 after tGCI. Collectively, our data demonstrated that reduced extracellular glutamate induced by HPC against tGCI through preventing the reduction of GLT-1 expression and maintaining GS activity in hippocampal CA1, which was mediated by upregulating Cx43 expression and inhibiting c-Src activity.